J. Biosci., Vol. 20, Number 4, September 1995, pp 525 530. Printed in India. Lipid phase transition in the plasma membrane of the goat epididymal maturing spermatozoa AJAY Ρ S RANA and GOPAL C MAJUMDER** Department of Sperm Biology, Indian Institute of Chemical Biology, 4, Raja S C Mullick Road, Jadavpur, Calcutta 700 032, India *Present Address: Diabetes Research Unit, Massachusetts General Hospital, MGH East Bldg. 149, 13th Street, Charlestown, ΜA 02129, USA MS received 16 November 1994; revised 28 June 1995 Abstract. Microviscosity of the highly purified plasma membranes isolated from the maturing goat caput, corpus and cauda epididymal sperm, was measured using l,6-diphenyl-l,3,5-hexatriene as the lipophilic probe at varying temperatures (12 42 C). As shown by the Arrhenius plot of the data each of the maturing sperm membranes had two distinct lipid phase transitions in the temperature zones 19 25 C and 34 37 C. The lowtemperature transitions for the immature caput- and mature cauda-sperm membranes were noted at 19 20 C, and 24 25 C, respectively, whereas both these membranes showed high temperature transition at 36 37 C. The maturing corpus-sperm membrane had phase transitions at 21 22 C and 35 36 C that were significantly different from those of the immature/mature sperm membranes. The data implicate significant alteration of the sperm membrane structure during epididymal maturation. The phase transition of the mature male gametes at 36 37 C may have a great impact on the subsequent events of the sperm life cycle since the mature spermatozoa that are stored in the epididymis a few degrees below the body temperature, experience higher temperature when ejaculated into the female reproductive tract. Keywords. Membrane fluidity; lipid phase transition; sperm maturation; epididymis; goat spermatozoa. 1. Introduction Testicular spermatozoa undergo maturation by some unknown mechanisms during transit through the epididymis when they acquire forward motility and fertility. The sperm surface is subjected to a variety of alterations during epididymal maturation although little is known about the precise identity of the cell surface molecules that may regulate the sperm maturation process (Yanagimachi 1981; Hammerstedt and Parks 1987). Lipid constituents of the plasma membrane (PM) have a pivotal role in determining its fluidity which in turn greatly influences the structure and function of the biomembranes (Stubbs and Smith 1984). PM lipids of the ram (Parks and Hammerstedt 1985), boar (Nikolopoulou et al 1985) and goat (Rana et al 1991) spermatozoa have been shown to undergo marked changes during epididymal maturation, suggesting thereby that the cell surface lipids may have an important role in modulating sperm maturation. Changes in sperm PM viscosity have been mostly studied with the isolated membranes of specific domains of the sperm surface for determining regional lipid **Corresponding author. Abbreviations used: PM, Plasma membrane; DPH, l,6-diphenyl-l,3,5-hexatriene. 525
526 Ajay Ρ S Rana and Gopal C Majumder variations (Vijayasarathy et al 1982; Vijayasarathy and Balaram 1982; Canvin and Buhr 1989). Recent studies using pyrene and l,6-diphenyl-l,3,5-hexatriene (DPH) as the lipophilic probes, have shown that the fluidity of the isolated goat sperm PM decreases significantly during the epididymal transit of sperm (Rana and Majumder 1990). Membrane fluidity is largely determined by cholesterol/phospholipid and saturated/unsaturated fatty acid ratios of the lipids (Stubbs and Smith 1984). The observed increase in the above-mentioned ratios of lipids and fatty acids during sperm maturation (Parks and Hammerstedt 1985; Rana et al 1991) appear to be primarily responsible for the maturation-dependent decrease of membrane fluidity. The data on the alteration of fluidity of the maturing goat sperm PM indicate that the epididymal maturity is associated with significant changes in the sperm PM structure which may be a prerequisite for the expression of motility and fertility characteristics (Rana and Majumder 1990). This study reports for the first time temperature-dependent lipid phase transitions in the isolated PM from the epididymal maturing sperm using goat as the model. 2. Materials and methods 2.1 Materials Polyethylene glycol compound (15,000 20,000 kda), Ficoll-400, dextran (average 229 kda) and DPH were obtained from Sigma Chemical Company, St. Louis, Mo, USA. Spermatozoa were extracted from fresh goat epididymides within 2 4 h after slaughter of the animals in the local slaughter houses. 2.2 Isolation of spermatozoa and plasma membrane Maturing goat epididymal spermatozoa were isolated from the caput, corpus and cauda segments of the epididymis as described earlier (Rana and Majumder 1987; Haldar et al 1990). The sperm were suspended in a modified Ringer's solution (RPS medium: 119 mm NaCl, 5mM KCl, 1 2 mm MgSO 4, 10 mm glucose, 16 3 mm K-phosphate, ph 6 9 and 50 U/ml penicillin). The corpus- and cauda-epididymal sperm were sedimented by centrifugation at 500 g for 5 min and then washed in RPS medium. The caput-epididymal immature sperm were isolated by a discontinuous Ficoll-400 density gradient centrifugation method (Haldar et al 1990). Highly purified plasma membrane was obtained from the cauda-epididymal mature sperm by using an aqueous two-phase polymer method (Rana and Majumder 1987). The isolated spermatozoa were subjected to hypotonic shock with 1 25 mm EDTA to dissociate the PM prior to dispersion of these cells to the polymer system consisting of 5 5% dextran and 4 2% polyethlene glycol. The cell suspension was then centrifuged at 9700 g for 30 min when the two phases separated and the membrane sedimented at the interphase. Purified PM was isolated from the corpus and caput sperm by a modification of this method as described earlier (Rana and Majumder 1989). The purified membrane was suspended in 10 mm Tris-HCl, ph 7 4. The isolated membranes showed a high degree of purity as judged by phase contrast and electron microscopic studies and analyses of marker enzymes characteristic of cellular organelles (Rana and Majumder 1987, 1989). The PM
Lipid phase transition 527 preparations had little contamination of intact/broken sperm fragments, cytoplasmic droplets and debris from epididymal mincing. Analysis of the isolated PM for acrosin and cytochrome oxidase specific markers of acrosome and mitochondira respectively, showed that it had no appreciable contamination of acrosomal/mitochondrial membranes. Protein contents of the membrane preparations were estimated by the method of Lowry et al (1951) using bovine serum albumin as standard. 2.3 Estimation of membrane viscosity Isolated sperm membrane fluidity which is the inverse of viscosity, was estimated using DPH as the lipophilic probe as reported before (Rana and Majumder 1990). One ml of the PM preparation (0 2 mg protein/ml) was mixed with 1 ml of DPH (2 µμ) solution in 10 mm Tris-HCl, ph 7 4 and the mixture was incubated at 37 C for 2 h in the dark prior to polarization measurement in Perkin-Elmer MPF-44B fluorescence spectrophotometer at a specified constant temperature. Samples were excited at 365 nm and the emission intensities were measured at 430 nm through a polarizer oriented parallel and perpendicular to the plane of the excitation beam. The samples were corrected for light scattering. Fluorescence polarization (P) which is inversely proportional to the fluidity, was calculated from an equation (Shinitzky and Barenholz 1978). Polarization values for each membrane preparation, were measured at a series of temperatures from 42 C to 12 C and 12 C to 42 C. The polarization values were similar in both temperature ranges. Approximate viscosity (η) was calculated from the equation proposed by Shinitzky and Barenholz (1978): 3. Results and discussion Arrhenius plots of the apparent viscosity parameters are linear for lipid system of invariant phase and therefore abrupt deviations from linearity are indicative of phase transitions (Shinitzky and Barenholz 1978; Brasitus 1983). As shown in figure 1, each of the isolated sperm plasma membrane fractions from caput, corpus and cauda showed two distinct phase transitions. Two phase transition temperature zones were identified in epididymal sperm PM: low temperature (19 25 C) and high temperature (34 37 C) zones (figure 1). The lower transition temperatures for the caput/corpus/cauda sperm PM, were 19 20 C, 21 22 C and 24 25 C, respectively whereas the membranes of these cells showed high-temperature transition points at 36 37 C, 34 35 C and 36 37 C respectively. It is possible that the observed phase transitions may be due to denaturation of membrane-associated proteins. However, it seems unlikely because proteins are not expected to undergo any appreciable denaturation in the temperature range: 12 42 C. The data clearly show that the phase transition temperatures in the low-temperature zone, undergo marked alterations during the epididymal maturation process. Previous studies using a constant temperature (25 C) have shown that the fluidity of the goat sperm PM, decreases with the epididymal sperm maturity (Rana and Majumder 1990). Arrhenius plots of the data on microviscosity at varying temperatures
528 Ajay Ρ S Rana and Gopal C Majumder Figure 1. Arrhenius plot of the microviscosity of the isolated plasma membranes from the maturing goat epididymal caput ( ), corpus ( ) and cauda (О) spermatozoa. The polarization values were estimated using the temperature range 42 to 12 C. The data shown are the mean ± SEM of microviscosity values generated on three different membrane preparations from each sperm maturative stage. (figure 1), confirm and extend the earlier data (Rana and Majumder 1990). The difference in the microviscosity between the mature and immature sperm PM, is most pronounced in the temperature range 17 19 C (figure 1). Membrane microviscosity is greatly influenced by cholesterol/phospholipid and saturated/ unsaturated fatty acids ratio of the lipids (Stubbs and Smith 1984). Maturationdependent marked increase in the above-mentioned ratios of lipids and fatty acids in goat sperm membrane (Rana et al 1991), may thus be primarily responsible for the observed changes in the microviscosity and phase-transition temperatures during epididymal sperm maturation (figure 1). This view is strengthened by the finding that lipid composition of PM derived from ram, boar and goat spermatozoa undergoes marked alteration during epididymal transit (Parks and Hammerstedt 1985;
Lipid phase transition 529 Nikolopoulou et al 1985; Rana et al 1991). Sperm surface proteins are greatly altered during transit through the epididymis (Johnson 1975; Majumder et al 1990). It is thus possible that in addition to lipids, the cell-surface proteins may as well play a significant role in maturation-dependent alteration of membrane viscosity/phase transition. It is well documented that the plasma membrane of the intact viable sperm possesses multiple distinct domains, each with its own characteristic structure (Friend 1982; Johnson 1975). It is thus not surprising that the bulk goat sperm PM preparations revealed more than one phase transition temperatures. More complex phase transitions have previously been reported in the isolated PM of the ejaculated ram spermatozoa (Holt and North 1986). Using DPH as the membrane probe, phase transitions occur in regions of 17, 26 and 36 C in the ram sperm membrane. It is noteworthy that maturation-dependent phase transition is most pronounced in the low temperature zone 19 25 C (figure 1), although little is known about the biological significance of this surface phenomenon. It is possible that this phase transition may add to the stability of the sperm membrane during cryopreservation of these cells and during exposure of the testicles of the intact animals to extremely low environmental temperatures. An interesting feature of mammalian testis and epididymis is that, these organs remain a few degrees below the body temperature of the animals. Upon ejaculation into the female reproductive tract, spermatozoa experience an increase in the surrounding temperature (around 37 C). The observation that the lipid phase of the isolated PM derived from mature goat sperm (figure 1) and ejaculated ram sperm (Holt and North 1986) undergoes transition when the environmental temperature is increased to approx. 37 C, suggests that the PM of the intact ejaculated spermatozoa will experience lipid phase transition in the female reproductive tract. Changes in the lipid phase from a liquid-crystalline to a gel state or vice versa have been recognized as having profound effects upon membrane properties including their permeability and ability to undergo membrane fusion reaction (Karnovsky et al 1982). It is therefore, probable that the sperm membrane phase transition in the female reproductive tract may play an important regulatory role for the subsequent events of the sperm life cycle: capacitation, acrosomal reaction and fertilization. Acknowledgements A research fellowship offered to APSR by the Indian Council of Medical Research and Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged. The authors are grateful to Dr Μ Basu, Prof. Ρ Nandi of Jadavpur University, Calcutta and to Drs A Ghosh and S Misra of Bose Institute, Calcutta for helpful discussion and to Mr Joydev Chatterjee of Regional Sophisticated Instrument Centre, Calcutta, for his technical assistance. References Brasitus Τ A 1983 Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes; Biochim. Biophys. Acta 728 20 30 Canvin Α Τ and Buhr Μ Μ 1989 Effect of temperature on the fluidity of boar sperm membranes; J. Reprod. Fertil. 85 533 540
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